This invention relates to methods for the determination of endotoxin which use as their basis the endotoxin-activated enzymatic coagulation or block polymerization of certain proteins native to Limulus polyphemus blood cells. In particular, this invention relates to those methods in which the Limulus protein coagulation is followed by nephelometry.
The detection of endotoxin in solutions and in biological fluids has long been important in the economics of commercial production of therapeutic intravenous solutions, and in diagnosis and prognosis of human disease. This test has become an important factor in the cost of manufacturing parenteral solutions because the heretofore employed US Pharmacopeia method has required live rabbits and a complex and lengthy assay procedure.
Pyrogens of greatest commercial and disease importance are derived from gram negative bacteria. The terms "endotoxin", and "pyrogen" all refer to substances present in the cell walls of bacterial families such as Brucellaceae, Enterobacteriaceae, Pseudomonadaceae, and Spirillaceae. Striking examples of the role of these substances in disease are evident when it is realized that endotoxins are the primary cause of fever, diarrhea, and abortion in diseases such as typhoid, cholera, food poisoning, and Brucellosis. Development of endotoxemia from bacteria residing in the gastrointestinal tract is a prime cause of death in appendicitis or other conditions involving rupture of the colon.
Endotoxin assays using Limulus blood cell extracts or lystates have gained considerable acceptance. These assays make use of the phenomenon that an enzyme present in the Limulus lysate is activated by endotoxin, and that once activated the enzyme catalyzes a reaction with other lysate proteins to form a crosslinked or coagulated protein matrix or gel. Since the amount of gel is proportional to the amount of endotoxin originally present, endotoxin in unknown samples may be readily determined.
Originally, formation of a Limulus lysate clot after incubation for a fixed time in the presence of endotoxin was the signal used to measure endotoxin concentration. While this method is generally more sensitve than the US Pharmacopeia rabbit test and is simple to conduct, it is insensitive and subject to considerable variation and error. The test is insensitive because coagulation occurring at low endotoxin levels may be unapparent to visual examination. It is subject to error because of the difficulty in subjectively determining the point at which a clot occurs.
The sensitivity and reproducibility of the endotoxin assay have been improved by the use of various optical methods for detecting lysate precipitation. Some methods measure the reduction in the amount of light which is passed by a suspension of coagulated Limulus protein, i.e., they measure the absorbence or turbidity of the suspension. See for example U.S. Pat. Nos. 4,038,029 and 3,915,805, and Hollander et al., "Biochem, Med." 15: 28-33 (1976). Other workers have separated the coagulated protein from the reaction mixture, then chromogenically determined the total amount of coagulated protein. See the example in U.K. Patent Specification No. 1,499,846.
Another optical method for determining the extent of coagulation is nephelometry. This method is frequently described or suggested in other terms, e.g., determination of light scattering. For example, U.K. Patent Specification No. 1,499,846, U.S. Pat. No. 3,915,805 and Levin et al. ("Thromb. Diath. Haemorrh." 19: 186-197 [1968]) all speak in terms of Limulus protein coagulation detection by "light scattering". For the purposes of this application nephelometric methods for the detection of endotoxin are those in which a determination is made of the amount of light reflected by a solution or suspension of coagulated Limulus amebocyte protein at greater than 0.degree. and less than 180.degree. from the incident beam of light as a measure of the extent of coagulation of the protein by an endotoxin-activated Limulus amoebocyte enzyme.
Nephelometric methods have not found widespread acceptance in endotoxin detection. In part, this may be attributed to the poor results that have been reported to date when using this technique. The principal problems which are evident are those of sensitivity and reproducibility. Levin et al. (FIG. 8) reported no increase in light scattering of Limulus amebocyte lysate when contacted with 40 pg endotoxin/ml., and not until the endotoxin level was increased to 4000 pg/ml was a lethargic response noted. Similarly, U.S. Pat. No. 3,915,805 reported an endotoxin detection threshold of 500 pg/ml. Additionally, inspection of the specific results reported by this patentee discloses considerable irregularity in the plotted points, even at high endotoxin concentrations. While U.K. Patent Specification No. 1,499,846 and Hollander et al. disclose low endotoxin thresholds the results were obtained using optical density or turbidimetric procedures.
It is therefore an object of this invention to improve the sensitivity and reproducibility of nephelometric endotoxin assays.
It is an additional object of this invention to achieve the sensitivity of known endotoxin assays with a comparatively simple, inexpensive nephelometric procedure.
It is another object of this invention to provide compositions and nephelometer improvements to enhance the reproducibility and sensitivity of the prior nephelometric assays for endotoxin using Limulus protein.
These and other objects will be apparent from a consideration of this invention as a whole.